Bacillus anthracis is the bacterium that causes the disease anthrax. Concern over its use as a bioweapon has renewed interest in elucidating mechanisms of how it causes disease. B. anthracis promotes its growth in the host through the production of lethal (LT) and edema toxin (ET), and the synthesis of a protective sheath, the capsule. Though it is clear that toxin production is necessary for B. anthracis to cause disease, there is conflicting evidence on the mechanisms by which toxins benefit the bacteria within a host. A number of studies that analyzed the effects of toxins on isolated cells suggested that both LT and ET paralyze the host immune system's cell-to-cell communications, while other studies indicated that the toxins enhanced the immune response by increasing the movement of defense cells towards the infection while at the same time increasing their ability to consume and destroy bacteria. We hypothesize that these conflicting observations regarding cell movement and activation of the host immune response reflect differences in the presence and activity of toxins over the time course of infection and reflect the inability of previous experimental techniques to continuously observe B. anthracis-host interactions from initiation to resolution. To address this hypothesis the movement and activation of defense cells, called monocytes, will be tracked within a mouse, in real-time and space, by optical and radionuclide imaging. Simultaneously, light-producing bacteria will also be tracked, so that the choreography of bacterial growth and dissemination through the host relative to monocytes can be analyzed. To determine the effect of bacterial toxins on the immune response, bacteria that are deficient in toxin will be compared to toxin-producing bacteria, so that each of the toxins separate activity on monocytes can be established. We predict that the immune system will initially respond to dormant spores, but will subsequently be impaired when spores germinate and begin secreting toxins. The successful implementation of these studies will grant basic insight into the means by which pathogens manipulate the immune response to their advantage, potentially providing tools by which we may intern control the immune system when it runs awry, and will establish powerful technologies for analyzing dynamic immune function. PUBLIC HEALTH RELEVANCE: Successful implementation of the proposed studies will provide a definitive demonstration of the relevance of secreted toxins for Bacillus anthracis pathogenesis;a poorly understood pathogen with high social relevance. Application of the technologies developed herein need not be limited to B. anthracis;essentially any infectious agent could potentially be studied with these methods. Beyond the realm of infectious disease, this technology will also benefit the study of many inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, lupus, and multiple sclerosis.